1. Field of the Invention
The invention relates to LHRH antagonists having improved solubility properties, processes for the preparation of these compounds, medicaments in which these compounds are contained, and the use of the medicaments for the treatment of hormone-dependent tumours and hormone-influenced non-malignant disorders such as benign prostate hyperplasia (BPH) and endometriosis.
2. Background Information
The nomenclature used for the definition of the peptides agrees with that nomenclature explained by the IUPAC-IUB Commission on Biochemical Nomenclature (European J. Biochem. 1984, 138, 9-37), in which, in agreement with the conventional representation, the amino groups at the N terminus appear to the left and the carboxyl group at the C terminus appears to the right. The LH-RH antagonists such as the peptides according to the invention include naturally occurring and synthetic amino acids, the former including Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His. The abbreviations for the individual amino acid residues are based on the trivial names of the amino acids and are Ala=alanine, Arg=arginine, Gly=glycine, Leu=leucine, Lys=lysine, Pal(3)=3-(3-pyridyl)alanine, Nal(2)=3-(2-naphthyl)-alanine, Phe=phenylalanine, Cpa=4-chlorophenylalanine, Pro=proline, Ser=serine, Thr=threonine, Trp=tryptophan, Try=tyrosine and Sar=sarcosine. All amino acids described here originate from the L series, if not mentioned otherwise. For example, D-Nal(2) is the abbreviation for 3-(2-naphthyl)-D-alanine and Ser is the abbreviation for L-serine. Substitutions on the xcex5 amino group in the side chain of lysine are represented by a term placed in brackets behind Lys, if appropriate in the form of an abbreviation.
Other abbreviations used are:
Ac Acetyl
Atz 3-Amino-1,2,4-triazole-5-carbonyl
B 4-(4-Amidinophenyl)amino-1,4-dioxobutyl
Boc tert-Butyloxycarbonyl
Bop Benzotriazol-1-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate
DCC Dicyclohexylcarbodiimide
DCM Dichloromethane
Ddz Dimethoxyphenyl-dimethylmethylenoxy-carbonyl (Dimethoxy-dimethyl-Z)
DIC Diisopropylcarbodiimide
DIPEA N,N-Diisopropylethylamine
DMF Dimethylformamide
Fmoc Fluorenylmethyloxycarbonyl
HF Hydrofluoric acid
HOBt 1-Hydroxybenzotriazole
HPLC High-pressure liquid chromatography
Me Methyl
TFA Trifluoroacetic acid
Z Benzyloxycarbonyl
The peptides according to the invention are analogues of the luteinizing -hormone-releasing hormone (LH-RH), which has the following structure:
p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, [LH-RH, gonadorelin] (SEQ ID NO:1).
For more than 20 years, researchers have sought selective potent antagonists of the LH-RH decapeptide [M. Karten and J. E. Rivier, Endocrine Reviews 7, 44-66 (1986)]. The high interest in such antagonists is based on their usefulness in the field of endocrinology, gynaecology, contraception and cancer. A large number of compounds have been prepared as potential LH-RH antagonists. The most interesting compounds which have been found to date are those compounds whose structures are a modification of the LH-RH structure.
The first series of potent antagonists was obtained by the introduction of aromatic amino acid residues into the positions 1, 2, 3 and 6 or 2, 3 and 6. The customary way of writing the compounds is as follows: the amino acids are first indicated which have taken the place of the amino acids originally present in the peptide chain of LH-RH, the positions in which the exchange took place being marked by superscripted figures. Furthermore, by the notation xe2x80x9cLH-RHxe2x80x9d placed afterwards it is expressed that these are LH-RH analogues in which the exchange has taken place.
Known antagonists are: [Ac-D-Cpa1,2, D-Trp3,6] LH-RH (D. H. Coy et al., In: Gross, E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6th American Peptide Symposium, pp. 775-779, Pierce Chem. Co., Rockville Ill. (1979): [Ac-Pro1, D-Cpa2, D-Nal(2)3,6] LH-RH (U.S. Pat. No. 4,419,347) and [Ac-Pro1, D-Cpa2, D-Trp3,6] LH-RH (J. L. Pineda, et al., J. Clin. Endocrinol. Metab. 56, 420, 1983).
In order to improve the action of antagonists, basic amino acids, for example D-Arg, were later introduced into the 6 position. For example [Ac-D-Cpa1,2, D-Trp3, D-Arg6, D-Ala10] LH-RH (ORG-30276) (D. H. Coy, et al., Endocrinology 100, 1445, 1982); and [Ac-D-Nal(2)1, D-Phe(4-F)2, D-Trp3, D-Arg6] LH-RH (ORF 18260) (J. E. Rivier et al., in: Vickery B. H. Nestor, Jr. J. J., Hafez, E.S.E (Eds). LHRH and its Analogs, pp. 11-22 MTP Press, Lancaster, UK 1984).
Further potent LH-RH antagonists are described in WO 92/19651, WO 94/19370, WO 92/17025, WO 94/14841, WO 94/13313, U.S. Pat. No. 5,300,492, U.S. Pat. No. 5,140,009, EP 0 413 209 A1 and DE 195 44 212 A1.
The latter discloses compounds having a modified ornithine or lysine unit in position 6 and which correspond to the following formula:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-Tyr5-D-Xxx6-Leu7-Arg8-Pro9-D-Ala10-NH2,
in which D-Xxx is an amino acid group of the general formula (VI) 
Further known LH-RH antagonists are antarelix, ganirelix and cetrorelix.
Antarelix:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-Tyr5-D-Hci6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2 
Ganirelix:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-Tyr5-D-hArg(Et)26-Leu7-hArg(Et)28-Pro9-D-Ala10-NH2 
Cetrorelix:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-Tyr5-D-Cit6-Leu7-Arg8-Pro9-D-Ala10-NH2.
The aim of the invention is to create novel LH-RH antagonists which have an increased enzymatic stability and significantly improved water solubility.
This object is achieved by compounds of the following general formula (I)
A-Xxx1-Xxx2-Xxx3-Xxx4-Xxx5-Xxx6-Xxx7-Xxx8-Xxx9-Xxx10-NH2xe2x80x83xe2x80x83(I)
in which
A is an acetyl or a 3-(4-fluorophenyl)propionyl group,
Xxx1 is D-Nal(1) or D-Nal(2),
Xxx2-Xxx3 is D-Cpa-D-Pal(3) or a single bond,
Xxx4 is Ser,
Xxx5 is N-Me-Tyr,
Xxx6 is D-Cit, D-Hci or a D-amino acid group of the general formula (II) 
in which n is the number 3 or 4, where R1 is a group having the general formula III
xe2x80x94(CH2)pxe2x80x94COxe2x80x94NR2R3xe2x80x83xe2x80x83(III)
where p is an integer from 1 to 4, R2 is hydrogen or an alkyl group and R3 is an unsubstituted or substituted aryl group or heteroaryl group, or R1 is a 3-amino-1,2,4-triazole-5-carbonyl group or R1 is a ring of the general formula (IV) 
in which q is the number 1 or 2, R4 is a hydrogen atom or an alkyl group, R5 is a hydrogen atom or an alkyl group and X is an oxygen or sulphur atom,
Xxx7 is Leu or Nle,
Xxx8 is Arg or Lys(iPr),
Xxx9 is Pro and
Xxx10 is Ala, D-Ala or Sar,
and their salts with pharmaceutically acceptable acids, in particular the acetates, embonates and trifluoroacetates.
Among the compounds according to the invention, those are particularly preferred in which Xxx6 is D-[xcex5-Nxe2x80x2-(imidazolidin-2-on-4-yl)formyl]-Lys, D-(3-amino-1,2,4-triazole-3-carbonyl)-Lys, abbreviated D-Lys(Atz) or D-[xcex5-Nxe2x80x2-4-(4-Amidinophenyl)-amino-1,4-dioxo-butyl]-Lys, abbreviated D-Lys(B).
Further particularly preferred compounds according to the invention are:
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Nle7-Arg8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(Atz)6-Leu7-Arg8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Leu7-Lys(iPr)8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Leu7 -Arg8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Nle7-Lys(iPr)8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Nle7-Lys(iPr)8-Pro9-Sar10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Hci6-Nle7-Arg8-Pro9-Sar10-NH2,
3-(4-Fluorophenyl)propionyl-D-Nal(1)1-Ser4-N-Me-Tyr5-D-Lys(Atz)6-Leu7-Arg8-Pro9-D-Ala10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7-Arg8-Pro9-Sar10-NH2,
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7-Arg8-Pro9-D-Ala10-NH2 and
Ac-D-Nal(2)1-D-Cpa2-D-Pal(3)3-Ser4-N-Me-Tyr5-D-Lys(B)6-Nle7-Lys(iPr)8-Pro9-Sar10-NH2 and also their salts with the abovementioned pharmaceutically acceptable acids.
The compounds according to the invention can be used for the treatment of hormone-dependent tumours, in particular prostate carcinoma or breast cancer, and also for non-malignant indications whose treatment necessitates LH-RH hormone suppression. For this, they are mixed with the customary vehicles and excipients and formulated as medicaments.
The synthesis of compounds according to formula (I) can both be carried out either by classical fragment condensation or by solid-phase synthesis according to Merrifield with synthesis following one another using D-lysine already acylated in the side chain with the carboxylic acid of the general formula R1xe2x80x94COOH or by reaction of a decapeptide unit with the appropriate carboxylic acids by amide linkage in the side chain of D-lysine6. Accordingly, the introduction of the R1xe2x80x94COxe2x80x94 group can be performed in three different positions in the process: before the condensation of the individual units to give the peptide, after the incorporation of lysine or ornithine in the peptide chain, but before the condensation of the next unit or after condensation of all units.
The compounds of the formula (I) are synthesized according to the known methods, such as, for example, by pure solid-phase technique, partly solid-phase technique (so-called fragment condensation) or by the classical solution couplings (see M. Bodanszky, xe2x80x9cPrinciples of Peptide Synthesisxe2x80x9d, Springer Verlag 1984).
For example, the methods of solid-phase synthesis are described in the textbook xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d, J. M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, Ill, 1984, and in G. Barany and R. B. Merrifield xe2x80x9cThe Peptidesxe2x80x9d, Ch. 1, pp. 1-285, 1979, Academic Press Inc. Classical solution syntheses are described in detail in the treatment xe2x80x9cMethoden der organischen Chemie [Methods of Organic Chemistry] (Houben-Weyl), Synthese von Peptidenxe2x80x9d[Synthesis of Peptides] E. Wunsch (Editor) 1974, Georg Thieme Verlag, Stuttgart, FRG.
The stepwise synthesis is carried out, for example, by first covalently bonding the carboxy-terminal amino acid whose xcex1-amino group is protected to an insoluble support which is customary for this, removing the xcex1-amino protective group of this amino acid, bonding the free amino group thus obtained to the next protected amino acid via its carboxyl group, and in this manner linking the customary amino acids of the peptide to be synthesized in the correct sequence step for step, and after linkage of all amino acids removing the finished peptide from the support and removing any further side function protective groups which may be present. The stepwise condensation is carried out in a conventional manner by synthesis from the corresponding, customarily protected amino acids.
The linkage of the individual amino acids to one another is carried out according to the methods customary for this; those particularly suitable are:
Symmetrical anhydride method in the presence of dicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC, DIC)
Carbodiimide method generally
Carbodiimide/hydroxybenzotriazole method (see The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer).
In the fragment coupling, the azide coupling, which proceeds without racemization, or the DCC-1-hydroxybenzotriazole or DCC-3-hydroxy-4-oxo-3,4-dihyro-1,2,3-benzotriazine method is preferably used. Activated esters of fragments can also be employed.
Esters of N-protected amino acids, such as, for example, N-hydroxysuccinimide esters or 2,4,5-trichlorophenyl esters, are particularly highly suitable for the stepwise condensation of amino acids. The aminolysis can be very well catalysed by N-hydroxy compounds which have approximately the acidity of acetic acid, such as, for example, 1-hydroxybenzotriazole.
Intermediate amino protective groups which present themselves are groups which are removed by hydrogenation, such as, for example, the benzyloxycarbonyl radical (=Z radical) or groups which can be removed by weak acid. Suitable protective groups for the xcex1-amino groups are, for example: tertiary butyloxycarbonyl groups, fluorenylmethyl-oxycarbonyl groups, carbobenzoxy groups or carbobenzothio groups (if appropriate in each case having a p-bromo- or p-nitrobenzyl radical), the trifluoroacetyl group, the phthalyl radical, the o-nitrophenoxyacetyl group, the trityl group, the p-toluenesulphonyl group, the benzyl group, benzyl radicals substituted in the benzene nucleus (p-bromo-or p-nitrobenzyl radical) and the xcex1-phenylethyl radical. Reference is also made here to P. Greenstein and Milton Winitz, Chemistry of Amino Acids, New York 1961, John Wiley and Sons, Inc., Volume 2, for example page 883 et seq., xe2x80x9cPrinciples of Ppetide Synthesisxe2x80x9d, Springer Verlag 1984, xe2x80x9cSolid Phase Peptide Synthesisxe2x80x9d, J. M. Stewart and J. D. Young, Pierce Chem. Company, Rockford, Ill., 1984, G. Barany and R. B. Merrifield xe2x80x9cThe Peptidesxe2x80x9d, Ch. 1, pp. 1-285, 1979, Academic Press Inc., and also The Peptides, Volume 2, Ed. E. Gross and J. Maienhofer, Academic Press, New York. These protective groups are fundamentally also suitable for the protection of further functional side groups (OH groups, NH2 groups) of the corresponding amino acids.
Hydroxyl groups present (serine, threonine) are preferably protected by benzyl groups and similar groups. Further amino groups not in the xcex1-position (for example amino groups in the xcfx89-position, guanidino group of arginine) are preferably orthogonally protected.
The individual amino acid units, excluding lysine or ornithine modified by the R1xe2x80x94COxe2x80x94group, are commercially obtainable. A possible course of the process for the preparation of the latter compounds is as follows:
1. The xcex1-carboxylic acid group is amidated.
2. The xcex5-amino group is protected by the Z group.
3. The xcex1-amino group is protected by the Boc group such that a selectivity with respect to the later removal of the amino protective groups results.
4. The Z group on the xcex5-amino group is removed.
5. The desired group R4xe2x80x94COxe2x80x94 is introduced on the xcex5-amino group.
6. The Boc group on the xcex1-amino group is removed.
7. The xcex1-amino group is provided with the Z group.
For the introduction of the R1xe2x80x94COxe2x80x94group by reaction of the amino group of the lysine with appropriate carboxylic acid, suitable processes are fundamentally the same processes as described above for the linkage of the amino acids. However, condensation using carbodiimide, for example 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide, and 1-hydroxybenzotriazole is particularly preferred.
The reaction for the linkage of the amino acids takes place in an inert solvent or suspending agent which is customary for this (for example dichloromethane), it being possible to add dimethylformamide, if necessary, to improve the solubility.
Suitable synthetic supports are insoluble polymers, for example polystyrene resin in bead form, which can be swollen in organic solvents (for example a copolymer of polystyrene and 1% divinylbenzene) . The synthesis of a protected decapeptide amide on a methylbenzhydrylamine resin (MBHA resin, i.e. polystyrene resin provided with methylbenzhydrylamine groups), which affords the desired C-terminal amide function of the peptide after HF cleavage from the support, can be carried out according to the following flow diagram:
Peptide synthesis protocol
The Nxcex1-Boc-protected amino acids are customarily coupled in a three fold molar excess in the presence of diisopropylcarbodiimide (DIC) and 1-hydroxybenzo-triazole (HOBt) in CH2Cl2/DMF in the course of 90 min, and the Boc-protected group is removed by action of 50% trifluoroacetic acid (TFA) in CH2Cl2 for half an hour. To check for complete conversion, the chloranil test according to Christensen and the Kaiser""s ninhydrin test can be used. Radicals of free amino functions are blocked by acetylation in a five fold excess of acetylimidazole in CH2Cl2. The sequence of the reaction steps of the peptide synthesis on the resin follows from the flow diagram. For the removal of the resin-bound peptides, the respective final product of the solid phase synthesis is dried in vacuo over P2O5 and treated at 0xc2x0 C. for 60 min in a 500-fold excess of HF/anisole 10:1/v:v.
After distilling of HF and anisole in vacuo, the peptide amides are obtained as white solids by washing with anhydrous ethyl ether with stirring, and the removal of polymeric support additionally obtained is carried out by washing with 50% strength aqueous acetic acid. By careful concentration of the acetic acid solutions in vacuo, the respective peptides can be obtained as highly viscous oils, which are converted into white solids after addition of abs. ether in the cold.
Further purification is carried out by routine methods of preparative high-pressure liquid chromatography (HPLC).
The conversion of the peptides into their acid addition salts can be effected in a manner known per se by reaction thereof with acids. Conversely, free peptides can be obtained by reaction of their acid addition salts with bases. Peptide embonates can be prepared by reaction of trifluoroacetic acid salts (TFA salts) of the peptide with free embonic acid (pamoic acid) or the corresponding disodium salt of embonic acid. For this, the peptide TFA salt is treated in aqueous solution with the solution of disodium embonate in polar aprotic medium, preferably dimethylacetamide, and the pale yellow precipitate formed is isolated.